Extraction-free detection kit and detection method for oncogene mutation in human thyroid glands
A detection kit and technology for thyroid cancer, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of poor prognosis and high tumor invasiveness
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Embodiment 1
[0031] Example 1 The specific primer sequence for amplifying the mutation site of thyroid cancer-related genes was synthesized by Shanghai Sangong.
[0032] Primer sequence pairs for BRAF:
[0033] BRAF-F
[0034] 5'-GAACGACATGGCTACGATCCGACTTCAGTGGAAAAATAGCCTCAATTCTTACCA-3'
[0035] BRAF-R
[0036] 5'-TCCTAAGACCGCTTGGCCTCCGACTTCTTCATGAAGACCTCACAGTAAAAATAGGT-3'
[0037] Primer sequence pairs for TERT:
[0038] TERT_F 5'-GAACGACATGGCTACGATCCGACTTAGCGCTGCCTGAAACTCG-3'
[0039] TERT_R 5'-TCCTAAGACCGCTTGGCCTCCGACTTCCTTCACCTTCCAGCTCCG-3'
[0040] Primer sequence pairs for NRAS:
[0041] NRAS-F
[0042] 5'-GAACGACATGGCTACGATCCGACTTCAAATGACTTGCTATTATTGATGGCAA-3'
[0043] NRAS-R
[0044] 5'-TCCTAAGACCGCTTGGCCTCCGACTTGTTATAGATGGTGAAACCTGTTTGTTGG-3'
[0045] NRAS-F
[0046] 5'-GAACGACATGGCTACGATCCGACTTTGGGATCATATTCATCTACAAAGTGGTT-3'
[0047] NRAS-R
[0048] 5'-TCCTAAGACCGCTTGGCCTCCGACTTGATTACTGGTTTCCAACAGGTTCTTG-3'
[0049] Primer sequence pairs for HRAS:
[0050] HRAS-F 5...
Embodiment 2
[0084] Embodiment 2: the pretreatment of sample
[0085] Qualified professionals use a puncture needle to sample tissue. After the biopsy is completed, the tissue should be completely immersed in PBS as soon as possible. Before sample transportation, samples were stored in -20°C or -80°C refrigerators.
Embodiment 3
[0086] Embodiment 3: Construction of library
[0087] The library construction process of this kit is as follows:
[0088] (1) Treatment of punctured tissue samples with nucleic acid releasing agent
[0089] After the puncture tissue sample was taken out, centrifuged at 5000rpm for 3min, the supernatant was removed; 5μl of nucleic acid release agent (400mmol / L NaOH, 1mol / L formamide) was added to the pellet, mixed well, left at room temperature for 10 minutes, and then proceeded to One step experiment.
[0090] (2) Amplification of the target fragment
[0091] The composition of PCR reaction solution is 50mmol / L Tris-HCl, 30mmol / L KCl, 3.5mmol / L MgCl 2 , 600μmol / L dNTPs, 20pmol / L primers and 5U PCR hot start Taq enzyme.
[0092] Prepare the following system:
[0093]
[0094] Set the following conditions on the PCR instrument for the reaction:
[0095]
[0096] (3) The first round of PCR product purification
[0097] 1) After the programmed reaction, the sample wa...
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